This application claims priority under 35 U.S.C. xc2xa7119(a) to Chinese Application No. 00104350.1, entitled APPARATUS AND METHOD FOR HIGH THROUGHPUT ELECTROROTATION ANALYSIS, filed on Mar. 15, 2000 and Chinese Application No. 00124086.2, entitled APPARATUS AND METHOD FOR HIGH THROUGHPUT ELECTROROTATION ANALYSIS, filed on Aug. 18, 2000.
The present invention concerns the field of electrorotation and biophysics. More specifically, embodiments include micromachined or microfabricated electrorotation chips, which produce a rotating electric field and can evaluate the electrorotation behavior of biological and non-biological particles; an apparatus for analyzing the electrorotation properties of such particles; and methods of use thereof.
Modern biotechnological and pharmaceutical approaches use a variety of analytic techniques to ascertain the behavior or identity of a molecule, cell, or biological particle. For example, investigators frequently use microbead-based multiplexed assays for analyzing the types and concentrations of target molecules in an xe2x80x9cunknownxe2x80x9d solution (see, e.g., Fulton R. J. et al, Clinical Chemistry, 43:1749-1756, (1997)). The target molecules may include antigens, antibodies, oligonucleotides, receptors, peptides, or enzyme substrates and may be labeled with a reporting molecule (e.g., a fluorescent molecule). Typically, in such an assay, different types of microbeads are used. Each type of microbead can be distinguished from others based on physical and chemical properties such as color, size, fluorescence molecule types and fluorescence intensity. The surfaces of different types of microbeads containing different types of molecules, i.e., each type of microbead is surface-activated or coated with one type of molecule. The molecules coating the surface of the microbeads may also be antibodies, antigens, oligonucleotides, receptors, peptides, enzyme substrates. Ideally, each molecule coating the surface of the microbeads interacts with one class of target molecule in the xe2x80x9cunknownxe2x80x9d solution.
Next, the surface-coated microbeads of many types are mixed together and are incubated with the xe2x80x9cunknownxe2x80x9d solution to allow the target molecules from the xe2x80x9cunknownxe2x80x9d solution to interact with the immobilized molecules on the microbeads. Prior to the incubation, all the target molecules are pre-labeled with certain types of reporter molecules (e.g., fluorescent molecules). Following the incubation, the detection of the reporter bound to the different types of microbeads is performed. By one approach, flow cytometry is used to analyze the levels of fluorescence on the individual microbeads and also determine the type of microbead detected. Because a xe2x80x9cone-to-onexe2x80x9d correspondence between the labeled target molecule and the immobilized molecule can be made, the identity and concentration of the target molecule in the xe2x80x9cunknownxe2x80x9d solution can be determined. A high-throughput format is desirable for these assays to expedite the analysis.
Modern approaches to pharmaceutical development also involve the analysis of interactions between a target molecule and molecules, cells, or particles of interest. For example, cell-based techniques are frequently used to screen chemical compound libraries for new drugs. The screening process typically employs cells having a target molecule to which an interaction with a drug-candidate is sought. The target molecule-containing cells are loaded into different reaction wells and are exposed to chemical compounds from a compound library. The cells are incubated with the chemical compounds for a specified time and then are evaluated for an interaction between the chemical compound and the target molecule. The detection of the interaction can be accomplished in many ways and fluorescence-based systems are frequently used. A similar assay determines the response of specific cell types to various amounts of chemical compounds and times of exposure. This type of assay can also be performed with a fluorescence-based detection system but the experimental set up allows for the determination of quantitative information. Desirably, a high-throughput system is also used for these assays to expedite drug development. The present invention relates to biological analyses which utilize electrorotation to characterize the behavior or identity of a molecule, cell or particle. Electrorotation analyses involve observing the behavior of molecules, cells, and particles, as well as complexes of these materials in a electric field that is applied so as to cause a rotation of the studied material. xe2x80x9cElectrorotationxe2x80x9d is a term of art that refers to the rotation of a material in an electric field. When a material is subjected to a rotating field, it becomes electrically polarized and the induced polarizations interact with the applied rotating fields, which produces a rotating torque that drives the rotation of the material. The rotation behavior (e.g., rotation rate and direction) depends on the frequency of the rotating field and electrical properties that are specific for the material being rotated. The measurement of rotational behavior can be expressed as a function of the frequency of the applied rotating field. From these measurements, the electrical properties (e.g., electrical conductivity and permittivity) unique to the material can be derived.
A number of electrorotation-based techniques have been developed for distinguishing particle types and analyzing particle properties. For example, U.S. Pat. No. 4,626,506 discloses an approach to distinguish cells that are secreting a cellular substance (e.g., proteins, hormones, etc) from non-secreting cells in the suspension. Further, U.S. Pat. No. 4,634,669 discloses an approach to differentiate two groups of particles based on observing different rotating directions of the particles when the frequencies of the applied rotating field are varied. Still further, U.S. Pat. No. 4,801,543 discloses an approach to analyze different groups of particles based on exposing the particles to two superimposed, simultaneous rotating electrical field forces with opposite rotation directions. Additionally, International Publication No. WO 93/16383 discloses an approach for detecting target molecules, which involves forming a complex between micro-particles and a target species and observing the difference in electrorotation properties between the original micro-particles and the complexes.
More discussion of electrorotation and its uses can be found in, for example, Arnold and Zimmermann, Z. Naturforsch., 37c:908-915, (1982); Fuhr et al., Stud. Biophys. 108: 149-164, (1985); Gimsa et al., in Physical characterization of biological cells, Schutt W, Klinkmann H and Laprecht I and Wilson T editors, Gesundheit, Berlin, pp 295-323, (1991a); Huang et al., Phys. Med. Biol., 37: 1499-1517, (1992); Huang et al., Biochim. Biophys. Acta 1282:76-84, (1996); Wang et al., Biochim. Biophys. Acta. 1193: 330-344, (1994); Gascoyne, Becker FF and Wang, Bioelectrochem. Bioenerg. 36: 115-125, (1998); and Huang Y, et al., Biochim. Biophys. Acta 1417: 51-62 (1999).
The present invention concerns the manufacture and use of electrorotation chips and an apparatus for high-throughput analysis of many different types of particles. Advantageously, embodiments can be used for high-throughput analysis of the rotation behaviors of different particle types on a single electrorotation chip, which allows for a rapid determination of the electrical properties of many different particles. Embodiments can also be used for high-throughput analysis of molecule-molecule, molecule-particle, and particle-particle interactions. For example, the invention can be used for cell-based, high-throughput screening for potential drug molecules from a chemical compound library.
Embodiments include an electrorotation chip comprising a substrate and a plurality of electrorotation units disposed on said substrate, wherein each of said electrorotation units comprises a plurality of electrode elements that are positioned on said substrate such that a rotating electrical field is generated when a plurality of phase-shifted electrical signals are applied to said electrode elements. This electrorotation chip can have a substrate that comprises a material selected from the group consisting of a non-porous solid material, and a porous solid material. This non-porous solid material or said porous solid material can be selected from the group consisting of glass, silicon, plastic, and ceramic. Some embodiments also have electrorotation units that are distributed in a plurality of columns and rows. Other embodiments have a plurality of electrode elements that are positioned such that the rotating electrical fields generated by adjacent electrorotation units are in opposite directions. More embodiments have a plurality of electrode elements that are positioned such that an electrode element of a first electrorotation unit is electrically connected to the nearest electrode element of a second adjacent electrorotation unit.
The electrorotation chip described above can also have a plurality of electrode elements that are positioned such that an electrode element of a first electrorotation unit is also an electrode element of a second adjacent electrorotation unit. The electrorotation chip described above can further comprise at least one signal generator that generates the phase-shifted electrical signals, said at least one signal generator being electrically connected to said electrorotation units. This signal generator can generate a periodic waveform in some embodiments. In some embodiments, the phase-shifted electrical signals are generated using a plurality of analog filters to shift the phase of a signal. Further, in some embodiments N is the number of said electrode elements in each said electrorotation unit and the phase-shifted electrical signals have phase offset values of 0, 360/N, 360*2/N, 360*3/N, 360*(Nxe2x88x921)/N. The electrorotation chip described above can also comprise a plurality of switches that apply the phase-shifted electrical signals to said electrode elements when said switches are conducting. These switches can be selected from the group consisting of bipolar junction transistors and metal oxide semiconductor field-effect transistors.
In other embodiments, the electrode elements resemble a structure selected from the group consisting of a butterfly footprint, a rectangular shape, and one face shaped in an arc. The electrorotation chip can have electrorotation units that comprise at least three electrode elements uniformly disposed about a center of the rotating electric field. Some electrorotation chips have electrorotation units that comprise four electrode elements uniformly disposed about a center of the rotating electric field. Some electrorotation chips also have a plate joined to said electrorotation chip, wherein said plate comprises at least one hole. This plate can comprise a plurality of holes and, each of said holes can provide access to only one electrorotation unit.
Another aspect of the invention concerns an electrorotation device comprising a plurality of signal inputs each of said signal inputs receiving a signal which is shifted in phase from signals received by the other signal inputs, wherein each signal input is electrically connected to a plurality of electrode elements, said electrode elements being organized into a plurality of electrorotation units, each electrorotation unit comprising at least one electrode element electrically connected to each of said signal inputs, and wherein when said phase-shifted signals are applied to said electrode elements in said electrorotation units a rotating electric field is produced in said electrorotation units. This electrorotation device can be modified to have a plurality of electrode elements that are electrically connected to each signal input are electrically connected to one another. The plurality of electrode elements can also be positioned such that the rotating electrical fields generated by adjacent electrorotation units are in opposite or same directions. In many embodiments, the electrorotation device described above have electrorotation units that are disposed on a substrate. The electrode elements can be electrically connected to a signal input and can be electrically insulated from the electrode elements connected to other signal inputs.
In other embodiments, a plurality of electrode elements are electrically connected to one another and to each signal input by conductors, and the conductors between electrode elements, which are electrically insulted from one another, are distributed between at least two layers in said substrate. In some aspects of this electrorotation chip, the electrode elements are positioned such that an electrode element of a first electrorotation unit is also an electrode element of a second adjacent electrorotation unit. In other aspects of this embodiment, phase shifted signals provided by said signal generators may be selectively applied to said electrorotation units. This embodiment can also comprise switches that are disposed between said signal generators and said electrode elements such that said phase-shifted signals are applied to said electrode elements in said electrorotation units when said switches are conducting. These switches can be selected from the group consisting of bipolar transistors and metal-oxide-semiconductor-field-effect-transistors (MOSFETs). In some embodiments, a single signal generator generates said plurality of phase-shifted signals which are applied to said signal inputs and, in other embodiments, the phase difference between said plurality of phase-shifted signals is 360xc2x0/N where N is the number of electrode elements in each electrorotation unit.
Methods of the invention include, for example, a method of determining an electrical property of a particle comprising the steps of providing an electrorotation chip that comprises a substrate having a plurality of electrorotation units disposed thereon; placing at least one particle in said plurality of electrorotation units; inducing rotating electrical fields in said electrorotation units; and measuring the rotation of said at least one particle and thereby determining the electrical property of said at least one particle. In some aspects of this method, the particle is selected from the group consisting of a biological molecule, a biological complex, an immune complex, a liposome, a protoplast, a platelet, a virus, and a cell. hi other aspects, the rotation of said at least one particle is measured at more than one frequency. Desirably, the electrical properties of a plurality of particles are measured and said plurality of particles can be a heterogeneous population. The method above can further comprise identifying those particles that have similar electrical properties.
Another approach described herein involves a method of identifying an agent that changes the electrorotational properties of a cell comprising the steps of providing an electrorotation chip that comprises a substrate having a plurality of electrorotation units disposed thereon; placing at least one cell in said plurality of electrorotation units; contacting said cell with a candidate molecule; inducing rotating electrical fields in said electrorotation units; measuring the rotation of said cell; determining the presence or absence of an effect on the electrorotational properties of said cell by comparing the rotation of said cell before contact with said candidate molecule with the rotation of said cell after contact with said candidate molecule or by comparing the rotation of a control cell that was not exposed to said candidate molecule with the rotation of said cell after contact with said candidate molecule; and identifying said candidate molecule as an agent that changes said electrorotational properties of said cell if said cells contacted with said candidate molecule have different electrorotational properties than said cells before contact with said candidate molecule or said control cells. In some aspects of this method, rotation is measured at more than one frequency and the electrical property of a plurality of cells can also be measured. Additionally, the plurality of cells can be a heterogeneous population and the method can further comprise identifying those cells that have similar electrical properties.
Another method involves an approach to determine the identity or concentration of a molecule in a biological sample. Thus methode comprises the steps of providing an electrorotation chip that comprises a substrate having a plurality of electrorotation units disposed thereon; coating particles with a detection reagent which binds to said molecule; placing at least one coated particle in said plurality of electrorotation units; contacting said coated particles with said biological sample; applying an electrical signal to said electrorotation units; measuring the rotation of said coated particles; and determining the identity or concentration of said molecule in said biological sample by comparing the rotation of coated particles contacted with said biological sample with coated particles that have not been contacted with said biological sample. In some aspects of this method, the detection reagent is selected from the group consisting of a dye, an antibody, and a ligand.